The mosquito midgut is a critical barrier that Plasmodium parasites must overcome to complete their developmental cycle and be transmitted to a new vertebrate host. Previous confocal studies with fixed infected midguts showed that ookinetes traverse midgut epithelial cells and cause irreversible tissue damage. Here, we investigated the spatiotemporal dynamics of ookinete midgut traversal and the response of midgut cells to invasion. A novel mounting strategy was established, suitable fluorescent dye combinations were identified and protocols optimized to label mosquito tissues in vivo, and live imaging protocols using confocal microscopy were developed. Tracking data showed that ookinetes gliding on the midgut surface travel faster and farther than those that remain in the lumen or those that have invaded the epithelium. Image analysis confirmed that parasite invasion and cell traversal occur within a couple of minutes, while caspase activity in damaged cells, indicative of cellular apoptosis, and F-actin cytoskeletal rearrangements in cells extruded into the gut lumen persist for several hours. This temporal difference highlights the importance of hemocyte-mediated cellular immunity and the mosquito complement system to mount a coordinated and effective antiplasmodial response. This novel in vivo imaging protocol allowed us to continuously observe individual ookinetes in live mosquitoes within the gut lumen and during cell traversal and to capture the subsequent cellular responses to invasion in real time for several hours, without loss of tissue integrity.

中文翻译：

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疟原虫入侵蚊子中肠的活体成像。

蚊子中肠是疟原虫寄生虫必须克服的关键障碍，才能完成其发育周期并传播给新的脊椎动物宿主。先前对固定感染中肠的共聚焦研究表明，ookinetes 穿过中肠上皮细胞并造成不可逆的组织损伤。在这里，我们研究了 ookinete 中肠穿越的时空动态和中肠细胞对入侵的反应。建立了一种新的安装策略，确定了合适的荧光染料组合并优化了协议以在体内标记蚊子组织，并开发了使用共聚焦显微镜的实时成像协议。追踪数据显示，在中肠表面滑翔的ookinetes比那些留在腔内或侵入上皮的ookinetes移动得更快、更远。图像分析证实，寄生虫入侵和细胞穿越会在几分钟内发生，而受损细胞中的半胱天冬酶活性（指示细胞凋亡）和挤出到肠腔中的细胞中的 F-肌动蛋白细胞骨架重排持续数小时。这种时间差异突出了血细胞介导的细胞免疫和蚊子补体系统对发起协调和有效的抗疟原虫反应的重要性。这部活体小说 成像协议使我们能够在肠道腔内和细胞穿越过程中连续观察活蚊子中的个体ookinetes，并实时捕获随后的细胞对入侵的反应数小时，而不会损失组织完整性。